Mmushi T MSc (Microbiology).pdf

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3.6. Isolation of bioactive compounds from acetone fraction of A. dimidiata Isolation of bioactive compounds from acetone fraction was performed through the use of bioassay-guided fractionation. The compounds observed as white bands on a pink background are active antibacterial compounds. From the bioutography results (Fig. 3.12 and Fig. 3.15) there were distinct bioactive bands on the bioautograms loaded with extracts of A. dimidiata, V. infausta and M. udanta. Fig. 3.22 indicates the phytoconstituents of the selected plant (A. dimidiata) used for isolation of bioactive compounds. A B Fig. 3.22. The components profiling of Apodytes dimidiata developed in 100% HEX (A) and 100% DCM (B). The TLC plates were sprayed with vanillin-sulphuric acid to indicate the best solvent system to be used in separation of extracts in column chromatography. 68
Of the two solvent systems used from Fig. 3.22 it shows that DCM was the best solvent for further isolation since it had a better separation (more distinct separated bands) than hexane. Figure 3.23 shows the phytochemical analysis results of the first column chromatography which was ran with 100 % DCM and slightly increasing polarity with methanol, whereas Fig. 3.24 indicates the corresponding bioautography of the fraction. The 100 % DCM and 90:10 DCM:MEOH fractions are showing antimycobacterium activity indicated by clear zones. Another column chromatography was run with hexane to further purify the active compounds in Fig. 3.24. The first fractions in Fig. 3.25 (F1 100%HEX – F5 95:5 HEX:ETAC) were pooled and recorded as compound 1. The corresponding Fig. 3.26 indicates the bioactive compounds and fractions F1- F3 85:15 HEX:ETAC were further targeted for purification since they had a different R f value from compound A. Figure 3.27 shows the phytochemical and bioassay results for the pooled fractions in fig. 3.25 (compound A) with less activity indicating reduced clear zone intensity. The active compounds from Fig.3.28 were further purified by running a column with 3:1 HEX:DCM (Fig. 3.29–Fig. 3.31). Figure 3.32 shows the pooled fractions which were purified and labelled as compound B. 69
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SCREENING, ISOLATION AND PURIFICATI
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DEDICATION I dedicate this work to
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TABLE OF CONTENTS Page Declaration
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1.7 Microbiology and pathogenicity
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6.2.1 Preparation of 1000 ml Middle
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LIST OF FIGURES Page Fig. 1.1. The
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Fig. 3.10. Fig. 3.11. Fig. 3.12. Fi
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Fig. 3.27. Fig. 3.28. Fig. 3.29. Fi
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ABSTRACT The leaves of fifteen plan
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CHAPTER ONE 1.1 Introduction Plants
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and R. erythropolis ATCC 4277strain
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Current therapeutic applications of
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agricultural (pesticides and herbic
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Fig. 1.3. The chemical structure of
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national pharmacopoeias, published
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Snyder and Kirkland (1979) tested t
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African medicinal plants. In certai
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Page 37 and 38: equal volume of sterile distilled w
Page 39 and 40: Fig. 1.6. Albizia gummifera (Lemmen
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Page 53 and 54: Rhodococcus erythropolis is an aero
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Page 59 and 60: phytochemical analysis (section 2.4
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Page 65 and 66: BEA EMW CEF DCM HEX ACE MET DCM HEX
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Page 75 and 76: Table 3.2.Average MIC (mg/ml) and t
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Page 83 and 84: BEA EMW CEF Fig. 3.19. Bioautograms
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Page 113 and 114: e that active compounds may be in v
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Page 119 and 120: Eloff, J.N. 1999a. Which extractant
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Page 123 and 124: Kemper, K.J. 1999. Longwood Herbal
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Page 131 and 132: CHAPTER SIX APPENDICES 6.1. Appendi
Page 133 and 134: 121 o C and 1 atm. For preparation
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